The single-cell transcriptomic atlas and RORA-mediated 3D epigenomic remodeling in driving corneal epithelial differentiation

Proper differentiation of corneal epithelial cells (CECs) from limbal stem/progenitor cells (LSCs) is required for maintenance of ocular homeostasis and clear vision. Here, using a single-cell transcriptomic atlas, we delineate the comprehensive and refined molecular regulatory dynamics during human CEC development and differentiation. We find that RORA is a CEC-specific molecular switch that initiates and drives LSCs to differentiate into mature CECs by activating PITX1. RORA dictates CEC differentiation by establishing CEC-specific enhancers and chromatin interactions between CEC gene promoters and distal regulatory elements. Conversely, RORA silences LSC-specific promoters and disrupts promoter-anchored chromatin loops to turn off LSC genes. Collectively, our work provides detailed and comprehensive insights into the transcriptional dynamics and RORA-mediated epigenetic remodeling underlying human corneal epithelial differentiation.

Line 646: While the t-test is commonly used for comparing two groups, it is not appropriate for multiple comparisons, as is the case in the current study.
Figure 1C: It shows no difference in the expression of LSGALS3 in the PCW18 cell group compared to the other groups at other times a few weeks earlier than PCW18.Could the authors clarify the basis for their assertion that LSGALS3 is expressed in this group?Figure 2C: The volcano plots aim to present the differential gene expression data.However, it is unclear which plots correspond to which genes.
Figure 2J, 3H: The manuscript utilizes the kNN-DREMI score as both the vertical and horizontal axes.While the kNN-DREMI score is recognized for measuring relationships between genes, its use in this manner might not be appropriate.
Figure 3E: The label on the vertical axis of the figure is marked as "ROR" when it appears that it should be labeled "RORA" to reflect the gene accurately.

Reviewer #3 (Remarks to the Author):
In the report, the authors performed analysis of previously published single cell RNA-seq data for development and adult cornea and identified RORA and PITX1 as key factors for promoting corneal epithelia cell (CEC) development from limbal stem cells (LCS).To test this prediction, over expression and knock down of RORA and PITX1 in primary LSC culture have been tested.Furthermore, CHIP-seq of RORA and PITX1 along with histone epigenetic marks and HiC experiments are used to investigate the mechanisms of gene regulation.The author's effort on experimentally testing the hypothesis is great and the results are very encouraging.The overall results are largely consistent with the informatic analysis from single cell data and the connection of RORA and PITX1 in CEC development is interesting.My specific comments are the following: 1.The authors reanalyzed previously published datasets.In figure 1A's UMAP, cells from PCW17 appear to segregate, forming the 'E_corneal superficial' cluster.This could be indicative of an uncorrected batch effect.Indeed, the RNA velocity analysis in figures 1I and 1K portrays potentially inconsistent trajectory to this cluster.Exploring alternative metaanalysis tools like scVI or Harmony might yield more coherent clustering.2. RORA and PITX1 don't exhibit high expression until later developmental stages and are predominantly expressed in terminally differentiated CECs.This would lead one to anticipate that these factors play roles in CEC terminal differentiation rather than as precursory factors transitioning LSC to CEC.How do the authors reconcile this seeming discrepancy?Is there any evidence of these genes being active in proliferating progenitor cells derived from LSC in the adult cornea? 3.While the overexpression of RORA and PITX1 clearly induces marker gene expression characteristic of CECs and decrease in LSC proliferation in culture.Given their roles as transcription factors, it's not unexpected for their overexpression to instigate a cascade of gene expression.Yet, it's crucial to establish if their overexpression truly facilitates the LSC to CEC developmental transition.Validating that cultured cells undergo accelerated CEC development and result in genuine CECs is essential.4. Given the inherent differences between in vitro and in vivo settings, corroborating the central hypothesis using animal models like mice or zebrafish, especially through gene knockouts or knockdowns during development, would be critical.

Reviewer #4 (Remarks to the Author):
This is an interesting study where the authors use a single-cell transcriptomic atlas to show the molecular dynamics during human corneal epithelial cell development and differentiation.They show the role of RORA and PITX1, primarily in silico in driving limbal stem/progenitor cells to differentiate into corneal epithelial cells.Overall the study is of interest and wellperformed.Comments are below.1) At the low level of resolution and image quality, it is challenging to determine colocalization of the limbal stem cell maker S100A2 and KRT15.The authors need to provide higher quality images at greater magnification.It is also necessary to perform quantification showing the degree of overlap between S100A2 and KRT15.
2) The authors claim that limbal stem cells express IFITM3; however, in the right panel there is no co-localization with a marker.The authors need to perform double immunofluorescence with a limbal stem cell marker to make this claim.
3) The authors should refrain from using short-hand terms.For example, the text states that the limbal stem cell markers KRT15 and KRT14 were used (line 115), whereas the figure states K12 and K15.It's confusing why the text refers to KRT14; however, the figure states K12?The authors should clarify this.There is no notation in the figure legend or text that K12 or K15 refer to KRT15 and KRT14.Thus the authors need to write out all gene names in full.The authors should also write out corneal epithelial cells and limbal stem/progenitor cells each time rather than CECs for people outside the field who are not familiar with these terms.Similarly they should write out overexpression rather than the term OE. 4) In the text the authors state that LSGALS3 was primarily expressed in the suprabasal and superficial layers of the limbus and cornea; however, in the figure uses the term LGALS3.I assume the authors are referring to LGALS3, and this appears to be a mistake in the text, which should be corrected.The authors need to provide a higher resolution image in Figure 1D adult for LGALS3, as it's not possible to make out the expression in the suprabasal and superficial layers at low resolution.5) The authors need to annotate all the figures of the corneas to show where the suprabasal, superficial, and limbal-corneal regions are.The is important to perform as we cannot tell the regions or corneal histology based on DAPI nuclear staining alone.6) The authors state that PITX1 is expressed in the differentiated limbal-corneal epithelial layer of adults; however, no co-staining is performed.This should be performed with markers.Similarly we cannot determine if there is co-localization of RORA and KRT3 at the low level magnification.Quantification should be performed on co-localization studies.7) The authors make the claim "In a word, we highlighted that RORA-PITX1 axis is necessary and sufficient to dictate corneal epithelial differentiation."Most of the studies are performed in silico with the exception of in vitro studies of limbal stem cells.The author need to perform conditional knockout in mouse of RORA-PITX1 to make the claim that the RORA-PITX1 axis is necessary and sufficient to dictate corneal epithelial differentiation.If they do not perform in vivo studies, they need to modify their claims to indicate that the work was performed in silico and in vitro and further validation in vivo needs to be performed.8) The authors need to provide more details in methods to make the study reproducible.This is critical.For example, the sequence for shRNAs needs to be provided.The methods for immunofluorescence staining is inadequate.It is critical to provide primary antibodies, suppliers, concentrations, lot numbers and all the details.It's not acceptable to write "experiments performed as previously described."Similarly more details for ChIP-Seq needs to be provided and it cannot be written "ChIP-Seq performed as previously described".The primary antibodies for ChIP-Seq need to be provided.It's important for studies to be reproducible.All the lot numbers for all reagents and concentrations need to be provided as the methods are very thin on details.The code for bulk RNA-seq data analysis needs to be provided and the raw data deposited in a site such as GEO.It's critical that the authors provide all the code used for all the computational analysis in the paper and to deposit all the data in a site such a GEO.This will allow other researchers to use and benefit from the data, which isn't possible in the current form.Overall this is an interesting study and I would be supportive of publication if the authors make the changes much of which revolve around making the data more transparent, code and raw data more accessible, and methods more detailed to improve reproducibility.
This study investigated the transcription dynamics of human limbal stem cells (LSCs) differentiation into corneal epithelial cells (CECs).By using the publicly available single cell RNA sequencing datasets, the authors successfully delineated a mechanism of RORA and PITX1 mediated histone posttranslational modification, which further regulated the 3D chromosomal interactions to control the expression of CEC genes.This manuscript is wellwritten with advanced data analyses, supported by some validation cellular experiments.Here are my major comments: 1. Line 68-69: Please also provide some examples about what other cell types also express these markers (KLF4, PAX6, EHF and FOXC1).
Thank you for the reviewer's valuable suggestions.We add a sentence in the main text (line 67-70): "they are not CEC-specific and are also expressed in basal, suprabasal and superficial layers of the limbal epithelium."This can be demonstrated in the indicated citations.
Thank you for the reviewer's valuable suggestions.We have corrected this error in the revised manuscript (line 107).Thank you for the reviewer's valuable suggestions.We add p-values of the GO terms in Fig 1g .Thank you for the reviewer's valuable suggestions.We have added two references (PAX6: Exp Eye Res, PMID:27818314 and J Cell Sci, PMID: 28202689 ) to support this claim in the revised manuscript (line 144).

Please indicate the locations of limbus and central cornea in
6. Line 141: Why only focus on these 3 hub genes but not others like SPARC, GPC3, CRABP2, RPS3, RPL6, EEF1A1 and MEG3?
Thank you for the reviewer's valuable suggestions.We have listed all the hub genes (PTN, CRABP2, BCAM, GPC3, SPARC, MEG3, WNT6, EEF1A1, RPL6 and RPS3) in the revised manuscript (line 151-152).Thank you for the reviewer's valuable suggestions.We have corrected this error in the revised manuscript (line 193).Thank you for the reviewer's valuable suggestions.In order to show the gene expression changes better, we changed the volcano plot (Fig. 2c) to a heatmap and showed RORA and PITX1 in Fig 2c.10.Line 261: It is very interesting to see proliferation, in addition to differentiation, was also affected in PITX1-OE and RORA-OE.The authors could discuss why cell proliferation is also affected.
Thank you for the reviewer's valuable suggestions.In discussion section of the revised manuscript, we discuss cellular proliferation as follows (line 429-434): "It is well-known that cell cycle withdraw is required for stem cell differentiation.Interestingly, RORA and PITX1 also downregulated the proliferation-related genes like ETS1 and HMGA2, resulting in reduced LSC proliferation.Thus, the inhibition of LSC proliferation induced by RORA and PITX1 is important for LSC differentiation." 11: One miner comment on line 417: The word "underly" should be written as "underlie".
Thank you for the reviewer's valuable suggestions.We have corrected this spelling mistake in the revised manuscript (line 444).
Reviewer #2 (Remarks to the Author): The manuscript presents an integrative study that combines a reanalysis of previously published single-cell data with new molecular biological techniques to explore differentiation in the corneal epithelium.This multi-faceted approach adds depth and breadth to the investigation and strengthens the study.However, there are some concerns in the manuscript (see specific points below): 1.Please discuss the relationship with previous reports in light of the current single-cell results, which have indicated that RUNX1, PAX6, and SMAD3 cooperatively interact with each other and establish core transcriptional regulatory circuits in limbal stem/progenitor cells.
Thank you for the reviewer's valuable suggestions.In the discussion section, we discuss the relationship of RORA and PITX1 with RUNX1, PAX6 and SMAD3 as follows (line 434-442): "Our previous study revealed that RUNX1, PAX6 and SMAD3 are expressed in both LSCs and CECs and play an indispensable role in corneal epithelial homeostasis 43 .Loss of them not only disrupted CEC differentiation, but also induced cellular fate transition by activating the epidermal genes 43 .In contrast, depletion of RORA and PITX1 only blocked CEC differentiation, but did not induce cellular fate switch.Thus, RUNX1, PAX6 and SMAD3 are required for corneal epithelial fate determination, while RORA-PITX1 axis is important to corneal epithelial differentiation." 2.Previous report revealed that RORA strongly inhibited iPS induction from human corneal epithelial cells, indicating that RORA has a potential role in maintaining differentiated corneal epithelial cells (Cell Rep. 2016 May  10;15(6):1359-68.(PMID: 27134177)).It would be engaging for the authors to discuss it.
Thank you for the reviewer's valuable suggestions.In the discussion section, we discuss it as follows (line 422-425): "Interestingly, a previous report suggested that RORA strongly inhibits the induction of induced pluripotent stem cells from human CECs 42 , which further indicates that RORA plays a key role in maintaining CEC differentiation state." We also cite the paper (Cell Rep, 2016, PMID: 27134177) here.

Figure 3L :
Figure3L: The authors claim that OE significantly rescues the expression suppression caused by SR3335, but statistical evidence does not support this assertion.

Fig S4 :
Fig S4: If the target sequence for each transcription factor or enhancer region is known, please add it to the chart.

Figure 5 :
Figure 5: In the upper right corner of Figure 5, there appears to be a plot that suggests the representation of TADs.However, this part of the figure is not discussed in either the figure legend or the main text.
Thank you for the reviewer's valuable suggestions.we indicate the locations of the limbus and central cornea in Fig 1d in the revised version.
4. In Fig 1G, please also show the p-values of these GO terms.

7.
Line 186: There are 21 TFs in Fig 2A, instead of 20 TFs as stated in the main texts.

9.
Again please indicate the locations of limbus and central cornea in Fig 2F.
Thank you for the reviewer's valuable suggestions.we indicate the locations of limbus and central cornea in Fig 2f in the revised version.

3.
It would be interesting to understand the cellular fate by showing what happens to cell proliferation-related or cell-cycle-related genes as the cells move toward the surface.